Method for pretreating lignocellulose by using acid-base mixture catalyst

ABSTRACT

The present invention relates to a method for pretreating lignocellulose by using an acid-base mixture catalyst. The method pretreats lignocellulose, by using a mixture catalyst of an acid and a base, so as not to pass through additional neutralization steps, and carries out pretreatment and simultaneous saccharification and fermentation through an identical single reactor process, thereby having an effect of producing ethanol in an excellent production yield from lignocellulosic biomass while simplifying the total process and reducing equipment costs and total processing costs.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Phase of International PatentApplication No. PCT/KR2015/004158, filed Apr. 27, 2015, which claimspriority to Korea Patent Application No. 10-2014-0050186, filed Apr. 25,2014, the disclosures of which are incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates to a method for pretreating cellulosicbiomass by using a mixture catalyst of an acid and a base inpretreatment for pretreatment, saccharification, and fermentation oflignocellulose in just one reactor (one-pot process).

2. Discussion of Related Art

In order to produce a biofuel from lignocellulose, recalcitrance oflignocellulose itself should be alleviated through suitablephysicochemical pretreatment. However, since a major pretreatmentprocess is performed at a high temperature and a pH in a range of astrong acid or a strong base, degradative products (furfural, etc.) aregenerated, which cause not only sugar loss but also the inhibition of ayeast fermentation process.

Meanwhile, a process for producing a biofuel from lignocellulose mainlyprogresses through pretreatment, solid/liquid separation, solidswashing, liquid detoxification, liquid neutralization, enzymatichydrolysis, and ethanol fermentation processes. Among these, unitprocesses for removal of inhibitory compounds such as solid/liquidseparation, solids washing, liquid detoxification, and liquidneutralization make total operating costs significantly increase.Therefore, there are needs for the development of a strain which istolerable to the inhibitor, or of a new process in which an inhibitor isnot produced.

As part of such efforts, consolidate bioprocessing (CBP) in which enzymeproduction and fermentation are performed in a single step using agenetically modified microorganism was suggested (Lynd et at.,Biotechnol. Prog., 1999, 15, pp. 777-793), but has not realized yet.Also, whole slurry fermentation in which saccharification andfermentation are performed without a solid/liquid separation process wassuggested (Jung et at., Bioresour. Technol., 2013, 132, pp. 109-114),but was again found inconvenient to additionally remove inhibitors usingactivated carbon, etc. In addition, a method for combining pretreatmentwith hydrolysis using an ionic liquid was suggested as well, but wasfound inconvenient in that an ionic liquid should be separated andrecovered after pretreatment due to costs and toxicity thereof, and aspart of some other integrated process, a simultaneous saccharificationand fermentation process itself is impossible (Shi et al., Green Chem.,2013, 15, pp. 2579-2589).

Therefore, the development of processing technology capable ofsimplifying the overall process, reducing total operating costs, andimproving the production yield of ethanol is being demanded.

SUMMARY OF THE INVENTION

The main objective of the present invention is to provide a method forpretreating lignocellulose by using an acid-base mixture catalyst inpretreatment in order to obtain ethanol from lignocellulose by carryingout pretreatment, saccharification, and fermentation process in onereactor.

Another object of the present invention is to provide a method forproducing ethanol from lignocellulosic biomass by using the modifiedpretreatment method aforementioned.

For achieving the objects, the present invention provides a method forpretreating lignocellulosic biomass comprising, reacting an acid-basemixture catalyst having a pH value of 1 to 4 with lignocellulosicbiomass.

The present invention also provides a method for producing ethanol fromlignocellulosic biomass comprising, carrying out simultaneoussaccharification and fermentation on a whole slurry obtained bypretreating the lignocellulosic biomass with an acid-base mixturecatalyst having a pH value of 1 to 4.

The present invention is effective for increasing saccharificationefficiency by pretreating lignocellulosic biomass with an acid-basemixture catalyst, and thereby improving the production yield of ethanol.

Also, the present invention is effective for simplifying the overallprocess and thereby significantly reducing total operating costs sincepretreatment, enzymatic hydrolysis, saccharification, and fermentationprocess of lignocellulosic biomass are performed in one reactor, andadditional steps such as milling of biomass, solid/liquid separationafter pretreatment, separated solids washing, separated liquiddetoxification, pH neutralization of a whole slurry and the like are notundergone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view illustrating a conventional process forproducing ethanol from lignocellulosic biomass and a process accordingto the present invention.

FIG. 2 shows effects of pretreatment of rice straw with an acid, a base,and an acid-base mixture catalyst, that is, 0.04 M HCl, 0.04 M HCl/0.01M NaCl, 0.01 M NaOH, 0.01 M NaOH/0.01 M NaCl, 0.01 M NaCl, and 0.04 MHCl/0.01 M NaOH.

FIG. 3 shows enzymatic digestibility in a pH range and a totalconcentration of an acid-base mixture catalyst upon pretreatment of ricestraw.

FIGS. 4A, 4B, 4C and 4D-FIG. 4A shows enzymatic digestibility obtainedthrough enzymatic hydrolysis after pretreatment of rice straw withmixture catalyst of HCl/NaOH at various mixing ratio, FIG. 4B showsenzymatic digestibility obtained through enzymatic hydrolysis afterpretreatment of rice straw with mixture catalyst of HCl/NaOH at varioustotal concentration, FIG. 4C shows enzymatic digestibility obtainedthrough enzymatic hydrolysis after pretreatment of rice straw withmixture catalyst of HCl/KOH at various mixing ratio, and FIG. 4D showsenzymatic digestibility obtained through enzymatic hydrolysis afterpretreatment of rice straw with mixture catalyst of HCl/NH₃ at variousmixing ratio.

FIGS. 5A, 5B and 5C-FIG. 5A shows enzymatic digestibility obtainedthrough enzymatic hydrolysis after pretreatment of rice straw withmixture catalyst of H₂SO₄/NaOH at various mixing ratio, FIG. 5B showsenzymatic digestibility obtained through enzymatic hydrolysis afterpretreatment of rice straw with mixture catalyst of H₂SO₄/KOH at variousmixing ratio, and FIG. 5C shows enzymatic digestibility obtained throughenzymatic hydrolysis after pretreatment of rice straw with mixturecatalyst of H₂SO₄/NH₃ at various mixing ratio.

FIGS. 6A, 6B and 6C-FIG. 6A shows enzymatic digestibility obtainedthrough enzymatic hydrolysis after pretreatment of rice straw withmixture catalyst of CH₃COOH/NaOH at various mixing ratio, FIG. 6B showsenzymatic digestibility obtained through enzymatic hydrolysis afterpretreatment of rice straw with mixture catalyst of CH₃COOH/KOH atvarious mixing ratio, and FIG. 6C shows enzymatic digestibility obtainedthrough enzymatic hydrolysis after pretreatment of rice straw withmixture catalyst of CH₃COOH/ NH₃ at various mixing ratio.

FIGS. 7A, 7B and 7C shows results of comparing a relationship withenzymatic digestibility in accordance with a change in constituentingredients of rice straw such as glucan (FIG. 7A), xylan (FIG. 7B), andlignin (FIG. 7C) using an acid-base mixture catalyst of the presentinvention.

FIG. 8 shows a comparison between enzymatic accessibility of rice strawwhich is pretreated using an acid-base mixture catalyst of the presentinvention or not pretreated.

FIG. 9 shows effects of pretreatment of rice straw using an acid-basemixture catalyst on enzymatic saccharification of the present invention.

FIG. 10 shows the production yield of ethanol by simultaneoussaccharification and fermentation of rice straw pretreated using anacid-base mixture catalyst of the present invention over time.

FIG. 11 is a schematic view illustrating a comparison of the mass yieldof ethanol from rice straw in accordance with the overall process of thepresent invention.

FIG. 12 shows effects of pretreatment of an oil palm frond under acidic,neutral, and basic conditions after the preparation of mixture catalystsbased on types of an acid and a base.

FIG. 13 shows enzymatic digestibility obtained by performing enzymatichydrolysis on a whole slurry after the pretreatment of 10% (w/v)substrate based on a pH range, a total concentration of an acid-basemixture catalyst, a pretreatment temperature (150, 170, and 190° C.),and a pretreatment time (45, 90, 360, 720, 1200, and 1440 seconds) in amini thermal reactor.

FIGS. 14A and 14B show enzymatic digestibility of pretreated sugarcanebagasse in accordance with a mixing molar ratio of an acid-base mixturecatalyst of the present invention (a final concentration is 0.1 M) (FIG.14A) and a concentration of an acid-base mixture catalyst (mixed at amolar ratio of an acid:a base=2: 1) FIG. 14(B).

FIGS. 15A, 15B, 15C and 15D show an insoluble solids recovery yield(FIG. 15A), a glucan recovery yield (FIG. 15B), a lignin removal rate(FIG. 15C), enzymatic digestibility (FIG. 15D) of sugarcane bagasseobtained through pretreatment and enzymatic hydrolysis in accordancewith the incubation time of an acid-base mixture catalyst of the presentinvention at normal temperature.

FIG. 16 shows enzymatic digestibility of sugarcane bagasse pretreatedusing an acid-base mixture catalyst, HCl, NaCl, a mixture catalyst ofHCl+0.033 M NaCl, and water.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Hereinafter, the composition of the present invention will be describedin detail.

The present invention relates to a method for pretreatinglignocellulosic biomass comprising, reacting an acid-base mixturecatalyst having a pH value of 1 to 4 with lignocellulosic biomass.

The acid-base mixture catalyst is prepared in an acidic pH range bymixing an acid and base at an appropriate molar ratio and is used forreaction with lignocellulosic biomass.

A type of the acid used for the acid-base mixture catalyst may includesulfuric acid, maleic acid, hydrochloric acid, nitric acid, phosphoricacid, carbonic acid, formic acid, acetic acid, hydrofluoric acid, oxalicacid, or citric acid, etc., but is not limited thereto.

A type of the base used for the acid-base mixture catalyst may includesodium hydroxide, potassium hydroxide, calcium hydroxide, bariumhydroxide, ammonium hydroxide, calcium carbonate, potassium carbonate,or ammonia, etc., but is not limited thereto.

The acid-base mixture catalyst may be used by ensuring that a totalconcentration becomes 0.01 to 1 M by mixing an acid and a base at amolar ratio upon pretreatment of lignocellulosic biomass in order tominimize the generation of an inhibitor which impedes saccharificationand fermentation after the pretreatment of lignocellulosic biomass. Bycausing a mixing ratio of the acid and base to become an acidiccondition, that is, a condition of pH 1 to 4, the pH range is similar tothat when pretreatment with an existing acid catalyst, but, according toone embodiment of the present invention, it is confirmed that, as inFIGS. 2, 7, and 10, a saccharification yield when pretreatment with themixture catalyst of an acid and a base is higher than when pretreatmentwith only an acid catalyst. Through such a result, it is thought to loseits own catalyst characteristics which an acid or a base has when anacid and a base are mixed, but as seen in the present invention, when aspecific mixing ratio and concentration are provided, it was found thatthe mixture catalyst of an acid and a base not only improves asaccharification yield in a range of pH 1 to 4 but also does not undergoadditional neutralization steps using a base because a pH within a wholeslurry after pretreatment corresponds to a weak acidic range.

In addition, the acid-base mixture catalyst is stable at normaltemperature, and therefore stability as a catalyst for pretreatment isensured. According to one embodiment, it was found that whenpretreatment is performed after an acid-base mixture catalyst isincubated at normal temperature for about 48 hours before pretreatmentof sugarcane bagasse, a saccharification yield is not affected at all,and characteristics as a catalyst for pretreatment do not change.

According to one embodiment of the present invention, when the acid-basemixture catalyst is used, the generation of an inhibitor such as aceticacid, furfural, hydroxymethyl furfural, and the like may be reduced.

The acid-base mixture catalyst may be treated in an amount of 4 to 20%(w/v) by 1 part by weight of lignocellulosic biomass. When an amount ofsolids loading is more than 20%, it is difficult to transfer materialand heat such that saccharification efficiency may decrease, and when anamount of solids loading is less than 4%, a concentration of a finalproduct may decrease due to a low loading amount.

Lignocellulosic biomass such as rice straw, giant miscanthus, sugarcanebagasse, corn byproducts, switchgrass, poplar, oil palm byproducts, oak,an energy crop and the like may be used as lignocellulosic biomass forpretreatment, but is not limited thereto. In addition, such pretreatmentmay have a similar effect on cellulosic biomass or industrial wastecomposed of cellulose, hemicellulose, lignin and the like.

According to one embodiment of the present invention, rice straw, oilpalm frond, sugarcane bagasse and the like may be used aslignocellulosic biomass, and the rice straw, oil palm fronds, orsugarcane bagasse may be dried and ground to a size of a few mm or lessusing a rotary-type mill, but is not limited thereto.

The pretreatment reaction may be performed at temperatures of 100 to200° C. for 60 seconds to 2 hours, and have an effect of simplifying theoverall process and reducing production costs because pH of reactantcorresponds to a weak acid by using the mixture catalyst of an acid anda base, and thus a neutralization process which is performed before asaccharification and fermentation process does not need to beadditionally carried out.

The present invention also relates to a method for preparing ethanolfrom lignocellulosic biomass comprising, carrying out simultaneoussaccharification and fermentation on a whole slurry obtained bypretreating lignocellulosic biomass with an acid-base mixture catalysthaving a pH value of 1 to 4.

Generally, chemical pretreatment and a saccharification process usingenzymes cost the most when using biomass which is renewable forestrywaste. Conventional technologies placed an emphasis on an increase inyield through these, and also, there was a disadvantage of substantiallyincreasing total operating costs because the overall process includesexisting unit processes, for example, milling of biomass, solid/liquidseparation after pretreatment, separated solids washing, separatedliquid detoxification, pH neutralization of a whole slurry and the like,all of which are complicated processes.

In contrast, the present invention has differentiated features from theconventional technologies in that first, an acid-base mixture catalystis used in pretreatment, and second, additional neutralization steps arenot undergone, and finally, the overall process is performed in onereactor.

First, in the case of using an acid-base mixture catalyst inpretreatment, conventionally a pretreatment step using acid or base isseparately performed due to inherent properties of an acid and basecatalyst, but in the present invention, an acid and a base are mixed atan appropriate molar ratio, an acid-base mixture catalyst is used underan acidic condition of pH 1 to 4 in pretreatment, and thus, although anabsolute amount of a used catalyst decreases, a wholesaccharification/fermentation yield may be maintained without change.That is, there is an advantage of increasing the output amount ofproducts based on the input amount of biomass.

Next, additional neutralization steps are not undergone. Aneutralization step is absolutely necessary for a subsequentsaccharification and fermentation process since existing catalysts areapplied in a strong acidic or strong basic range of a pH, and existingcatalysts cause not only sugar loss but also the inhibition of a yeastfermentation process by generating glycolysis products with the additionof additional bases or acids. Furthermore, such addition of bases oracids makes a whole process economically worse in terms of costs for theaddition or costs for removing a produced salt.

In contrast, in the present invention, an acid-base mixture catalyst(ranging from pH 1 to pH 4) is used in pretreatment and then it isconfirmed that a pH of a whole slurry is approximately in a weak acidicrange, and as a result, additional neutralizing agents do not need to beadded besides a buffer for cultivating microorganisms. Also, since thewhole slurry obtained through pretreatment is used for producingproducts, an existing solid/liquid separation process, solids washing, aliquid detoxification process, a liquid neutralization process may beomitted, which causes costs to be considerably reduced in the biofueland biorefinery industry.

Finally, since simultaneous saccharification and fermentation process onthe whole slurry obtained by pretreatment is performed in one reactor,costs required for process equipment may remarkably decrease.

A method for preparing ethanol from lignocellulosic biomass of thepresent invention includes performing simultaneous saccharification andfermentation on the whole slurry of lignocellulosic biomass pretreatedwith a mixture catalyst of an acid and a base to prepare ethanolaccording to a schematic view illustrating a process in FIG. 1.

Since a pH of the whole slurry is in a weak acidic range and thusadditional neutralization steps do not need to be undergone beforesaccharification, saccharification and fermentation on the whole slurrymay be directly performed.

Generally, a saccharification step and a fermentation step may beperformed through a separate hydrolysis and fermentation (SHF) processin which saccharification and fermentation are carried out in individualreactors, or through a simultaneous saccharification and fermentation(SSF) process in which saccharification and fermentation are carried outin one reactor at the same time. In the SSF process, since yeast mayremove glucose through a fermentation step and accumulation of sugars inthe reactor may be minimized as soon as glucose is produced, inhibitionof a final product shown in the SHF process may be prevented andenzymatic hydrolysis may be improved. Also, equipment costs may bereduced, costs may be reduced by a low input amount of enzymes, and acontamination problem may decrease due to ethanol present in thereactor. Furthermore, since simultaneous saccharification andfermentation could be directly carried out in the reactor in which apretreatment step is performed, one-pot process of the present inventionmay be realized. Therefore, the present invention uses simultaneoussaccharification and fermentation.

Enzymes used in the saccharification may include cellulase, α-amylase,glucoamylase, endoglucanase, xylanase, β-glucosidase, α-agarase,β-agarase I, β-agarase II, β-galactosidase, neoagarobiose hydrolase,neoagarotetraose hydrolase, neoagarohexaose hydrolase, α-neoagarobiosehydrolase, or mixtures or composites thereof, etc., but is not limitedthereto.

In addition, microorganisms used in fermentation may include, forexample, Saccharomyces cerevisiae, Klebsiella oxytoca P2, Brettanomycescurstersii, Saccharomyces uvzrun, Candida brassicae. Sarcina ventriculi,Zymomonas mobilis, Kluyveromyces marxianus IMB3, Clostridiumacetobutylicum, Clostridium beijerinckii, Kluyveromyces fragilis,Brettanomyces custersii, Clostriduim aurantibutylicum, and Clostridiumtetanomorphum, etc., but the present invention is not limited thereto.

Conditions for performing the simultaneous saccharification andfermentation are not particularly limited, and the reaction may beperformed while stirring for example, in a concentration of initialglucose ranging 2 to 30% (w/v) at temperatures of 25 to 40° C. at a pHof 4.0 to 8.0 at a speed of 50 to 250 rpm.

If necessary, those skilled in the art can perform other additionalsteps and/or processes, for example, a purification step in which afermented liquid obtained by the simultaneous saccharification andfermentation step is purified according to a method known in the art.

Hereinafter, the present invention will be described in detail accordingto exemplary embodiments. However, the following exemplary embodimentsare merely presented to exemplify the present invention, and the contentof the present invention is not limited to the following exemplaryembodiments.

EXAMPLE 1 Effects of Saccharification or Simultaneous Saccharificationand Fermentation in Accordance with Pretreatment of Rice Straw

Rice straw was used as a representative example of lignocellulose, andglucose and the production of ethanol through a fermentation processwere confirmed as representative examples of products.

First, in order to confirm effects of pretreatment with an acid-basemixture catalyst, a mixture catalyst mixed based on types of an acid anda base was prepared, wherein a mixing ratio (a pH condition) andconcentration of the mixture catalyst were optimized, and in order toconfirm reaction mechanisms of the acid-base mixture catalyst and adifference with only an existing acid or base catalyst, biomasscomposition and enzymatic digestibility were compared. In addition,enzymatic accessibility of biomass treated with the acid-base mixturecatalyst was confirmed. Finally, saccharification and fermentation wereperformed in a reactor including a whole slurry without additionalprocesses such as solid/liquid separation, separated solids washing,separated liquid detoxification, pH neutralization of a whole slurry andthe like on the whole slurry of biomass pretreated with the acid-basemixture catalyst, and the yield of ethanol was confirmed (referring toFIG. 1).

Rice straw was harvested from Yeonggwang (South Korea), washed, dried,and then cut into a range of 90 to 1000 μm using a high speed rotarycutter (MF 10 commercially available from IKA, Staufen, Germany).

Hydrochloric acid, sulfuric acid, acetic acid, sodium hydroxide,potassium hydroxide, ammonia, sodium chloride and the like were mixed atan appropriate ratio, were diluted to a desired concentration, and wereused in a pretreatment step. While 2 g of dried rice straw were soakedin 20 mL of a catalyst in a 100 mL container and then was reacted in amini thermal reactor at 190° C. for 2 minutes, tests were performedbased on various changes in a mixing ratio of an acid and a base, aconcentration of a catalyst and the like, wherein a pH was under anacidic condition, a neutral condition, an alkaline condition and aconcentration was in a range of 0 to 1 M.

After completion of pretreatment, an insoluble solid was recovered byfiltering a pretreated slurry using a filter cloth (pore size: 22 to 25μm, Calbiochem, La Jolla, Calif., USA) for composition analysis.Afterward, the pretreated insoluble solid was washed with about 1 L ofdistilled water until a pH value of a washing solution becomes 6 to 7,and then a washed solid was dried in a vacuum drying oven for 3 days ormore at 45° C.

Composition analysis of pretreated biomass (a whole slurry) or untreatedbiomass was measured using a NREL method (Sluiter A et at., LaboratoryAnalytical Procedure: Determination of Structural Carbohydrates andLignin in Biomass, National Renewable Energy Laboratory, Golden, Colo.2008) for carbohydrates and acid-insoluble lignin. Moisture content inbiomass was measured on the basis of the NREL method. In compositionalanalysis of carbohydrates, sugar content and enzymatic digestibility aredetermined using an ion exchange column (HPX-87P; commercially availablefrom Biorad, Hercules, Calif.) including Pb²⁺, and sugar detection wasperformed through a refractive index detector. For measuring inhibitorssuch as acetic acid, furfural, hydroxymethyl furfural (HMF) and thelike, a column (HPX-87H; commercially available from Biorad, Hercules,Calif.) including H⁺ was used, and also the measurement was performedthrough a refractive index detector. All analyses were repeated 3 times.

For measuring enzymatic digestibility, the insoluble solids washed afterpretreatment of rice straw or untreated rice straw itself was subjectedto enzymatic hydrolysis using Accellerase 1000 (commercially availablefrom Genencor, Rochester, N.Y.) which is in the same added amount as 7to 60 FPU of cellulase/g-glucan. A reaction mixture including biomasswashed after pretreatment with 1% glucan was stirred in a 0.05 M sodiumcitrate buffer solution (pH 4.8) at 50° C. at 200 rpm, while beingcultivated in a shaking incubator. Enzymatic digestibility representedby the theoretical maximum yield of glucose was determined as a ratio ofglucose (g) produced through hydrolysis to the total glucose (g) withininitial biomass used in enzymatic hydrolysis.

For investigating fermentation capability of pretreated biomass, asimultaneous saccharification and fermentation test was performed. Afermentation medium was composed of a 0.05 M citric acid buffer solution(pH 4.8), a 1% (w/v) yeast extract and 2% (w/v) peptone. A untreatedsubstrate or a pretreated and washed substrate was added by a finalglucan concentration of 3% (w/v) in a total 100 mL of a medium and awhole slurry of bundle pretreated and neutralized was added using afinal substrate concentration of 6% (w/v) in a 250 mL-flask with aneedle-pierced silicone stopper for a micro-aerobic condition andventing CO₂ produced during fermentation. After sterilization,fermentation was performed using pre-grown Saccharomyces cerevisiae D₅A(ATCC 200062) while stirring at 38° C. at 170 rpm.

For analyzing the accessibility of cellulase with respect to celluloseof pretreated rice straw, binding capacity was measured using Type Asurface-binding protein3 (CtCBD3) derived from Clostridium thermocellum.5 mg of untreated or pretreated biomass was incubated with an excessamount of BSA protein or CtCBD3 and a 50 mM phosphate buffer solution(pH 7) and then supernatant and pellet were separated by centrifugationafter 3.5 hours, and an amount of unbound protein was measured by theBradford method.

TABLE 1 Composition analysis of rice straw pretreated by using differentcatalysts acid-base mixture catalyst (0.04M HCl + 0.01M HCl NaOH NaCluntreated NaOH) (0.04M) (0.01M) (0.01M) Component from insoluble solids(g/100 g dry rice straws before pretreatment) insoluble — 55.6 ± 2.555.9 ± 2.6 75.5 ± 0.8 85.0 ± 3.9 solids recovery yield glucan 35.8 ± 1.532.3 ± 0.6 30.9 ± 0.3 33.7 ± 0.0 33.1 ± 0.4 xylan 10.5 ± 1.4  3.9 ± 0.5 2.9 ± 0.2 10.7 ± 0.2 10.5 ± 0.4 galactan  0.3 ± 0.3  1.5 ± 1.4  2.2 ±0.0  2.9 ± 0.0  3.4 ± 0.0 arabinan  3.1 ± 0.5  1.8 ± 0.0  1.7 ± 0.0  2.3± 0.0  2.6 ± 0.0 lignin 18.2 ± 1.3 11.0 ± 0.3 10.7 ± 0.0 10.8 ± 0.3 12.2± 0.2 Component from dissolved solids (g/100 g rice straw beforepretreatment) glucose —  4.9 ± 0.2  6.1 ± 0.0 0.5 ± 0.0 1.5 ± 0.0hemicellulose — 10.2 ± 0.1 14.9 ± 0.1 0.5 ± 0.0 1.3 ± 0.1 monomer ^(c)acetic acid —  1.5 ± 0.0  2.0 ± 0.1 1.6 ± 0.0 0.3 ± 0.1 HMF —  0.4 ± 0.0 0.5 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 furfural —  0.6 ± 0.0  0.7 ± 0.0 0.0 ±0.0 0.0 ± 0.0 ^(a) Pretreatment conditions: performed in a mini thermalreactor at 190° C. for 2 minutes, and solids loading in an amount of 10%(w/v) ^(b) Experimental data is expressed as means ± standard deviations^(c) Hemicellulose monomer includes xylose, galactose, and arabinose ina liquid fraction thereof

As shown in FIG. 2, after pretreatment with an acid, a base, or anacid-base mixture catalyst, the use of an acid-base mixture catalystproduces relatively excellent enzymatic digestibility.

FIG. 3 shows enzymatic digestibility in a pH range and a totalconcentration of an acid-base mixture catalyst when pretreating ricestraw, wherein a 10% (w/v) substrate was pretreated by loading in a minithermal reactor at 190° C. for 2 minutes and then enzymatic hydrolysiswas performed on biomass washed with distilled water, and stirring wasperformed with the addition of 15 FPU/g glucan at pH 4.8 at 50° C. at200 rpm for 50 hours. In FIG. 3, an acidic condition refers to a case inwhich an acidic proportion is high within a mixture catalyst, a neutralcondition refers to a case in which an acidic proportion is similar to abasic proportion, and a basic condition refers to a case in which abasic proportion is high, each of which means a pH range of about pH 1to 4, 4 to 9, and 8 to 14, respectively. Also, in FIG. 3 a concentration“0” means treatment with water, which is also applicable to FIGS. 4 and13.

This process results in confirming if the pretreatment was properlyperformed, wherein, in the case of pretreatment with the acid-basemixture catalyst, effects of pretreatment were improved under an acidiccondition (range of pH 1 to 4 in which an acidic proportion is high),and the more a concentration of the mixture catalyst increased, the moreeffects of pretreatment were improved (FIG. 3B).

FIGS. 4 to 6 show enzymatic digestibility obtained through enzymatichydrolysis after pretreatment of rice straw with mixture catalystsobtained by varying a type and mixing ratio of an acid and a base,wherein the mixture catalyst was obtained by mixing a 0.05 M acid and a0.05 M base at a molar ratio at 190° C., loading a 10% (w/v) substratein the mixture catalyst in a mini thermal reactor, pretreating withreaction at 190° C. for 2 minutes, and then performing enzymatichydrolysis using 15 FPU Accellerase 1000/g-glucan (while stirring at pH4.8 at 50° C. at 200 rpm for 50 hours).

As a result, mixture catalysts of a strong acid and a strong base showedsimilar patterns based on a mixing ratio, and mixture catalysts of aweak acid and a strong base showed no significant difference in asaccharification yield under an acidic, neutral, basic condition, whichindicates that there is the need for condition optimization.

FIG. 7 shows results of comparing a relationship between data obtainedthrough composition analysis after pretreatment with an acid-basemixture catalyst in an acidic range at 190° C. for 2 minutes and dataobtained through hydrolysis while stirring at 50° C. at 200 rpm for 50hours, wherein a correlation between glucan and a hydrolysis ratio was0.20, a correlation between xylan and a hydrolysis ratio was 0.95, and acorrelation between lignin and a hydrolysis ratio was 0.91, andtherefore common effects of xylan removal as an effect of an acidicsolution and lignin removal as an effect of a basic solution wereobserved.

FIG. 8 shows a comparison of enzymatic accessibility of rice straw whichis pretreated or not treated, wherein the binding reaction with ricestraw was performed using BSA (control group) and CtCBD3 in a 50 mMphosphate buffer solution at pH 7 at 4° C. for 3.5 hours, and as aresult, it was confirmed that accessibility with respect to cellulose isimproved.

FIG. 9 shows effects of pretreatment with an acid-base mixture catalyston enzymatic saccharification, wherein, when pretreatment was performed,a hydrolysis ratio at a concentration for treating cellulase was about60 to about 80%, but in the case of biomass which was not pretreated, ahydrolysis ratio was 20% or less.

FIG. 10 shows results of measuring the production yield of ethanol bysimultaneous saccharification and fermentation of pretreated biomass,wherein it can be seen that when a mixture catalyst of an acid and abase was used, ethanol yield is improved by as much as 17.5% than whenbiomass was pretreated with only an acid catalyst, and also the yield isimproved by as much as about 20% than when a washing process wasundergone after pretreatment with a mixture catalyst.

Finally, it was found that an additional 3.4 g of ethanol were producedthrough process integration after pretreatment based on 100 g of biomassunder the limitation that only glucose was used compared to whenindividual processes were performed separately (FIG. 11).

EXAMPLE 2 Investigation of Enzymatic Digestibility in Accordance withPretreatment of Oil Palm Frond

An oil palm frond was used as lignocellulose and was pretreated with anacid-base mixture catalyst, and then a saccharification yield wasmeasured through enzymatic hydrolysis.

In order to confirm effects of pretreatment with an acid-base mixturecatalyst, a mixture catalyst mixed based on types of an acid and a basewas prepared, wherein a mixing ratio (a pH condition) and concentrationof the mixture catalyst were optimized, and enzymatic digestibility wascompared. Conditions of pretreatment and a method for performingenzymatic hydrolysis are the same as in Example 1.

An oil palm frond was harvested, washed, dried, and then cut into arange of 90 to 1000 μm using a high speed rotary cutter (MF 10commercially available from IKA, Staufen, Germany).

FIG. 12 shows an obtained result of pretreatment effect of oil palmfrond under acidic, neutral, and basic conditions after the preparationof mixture catalysts based on types of an acid and a base, whereinindividual mixture catalysts show high enzymatic digestibility under anacidic condition, but in the case of sulfuric acid and sodium hydroxideor ammonia, the difference in a saccharification yield under acidic,neutral, and basic conditions was not significant. In this case, itseems that condition such as pretreatment conditions, a mixingconcentration and the like need to be optimized.

FIG. 13 shows enzymatic digestibility obtained by performing enzymatichydrolysis on a whole slurry with the addition of 15 FPU/g glucan andstirring at pH 4.8 at 50° C. at 200 rpm for 50 hours after thepreparation of a 10% (w/v) substrate based on a pH range and totalconcentration of an acid-base mixture catalyst, a pretreatmenttemperature (150, 170, and 190° C.), and a pretreatment time in a minithermal reactor (45, 90, 360, 720, 1200, and 1440 seconds), wherein, aswith the above-described rice straw, in the case of pretreatment with anacid-base mixture catalyst, the more a concentration of a mixturecatalyst increased under an acidic condition, the higher effects ofpretreatment were, and a pretreatment temperature was the most effectiveat 190° C. and effects were maintained when a reaction time is 90seconds or more.

EXAMPLE 3 Investigation of Saccharification Yield in Accordance withPretreatment of Sugarcane Bagasse

Sugarcane bagasse was used as lignocellulose, was pretreated with anacid-base mixture catalyst, and then a saccharification yield wasmeasured through enzymatic hydrolysis.

In the case of sugarcane bagasse, sugarcane was harvested, washed,dried, and then was cut into a range of 90 to 1000 μm using a high speedrotary cutter (MF 10 commercially available from IKA, Staufen, Germany).

In order to confirm effects of pretreatment with an acid-base mixturecatalyst, a mixing molar ratio (12 to 2:1=acidic; 1:1=neutral; 1:2 to 4:basic condition) and concentrations of an acid and a base wereoptimized, and enzymatic digestibility was compared. Conditions ofpretreatment and a method for performing enzymatic hydrolysis are thesame as in Example 1.

FIG. 14 shows enzymatic digestibility of pretreated and washed sugarcanebagasse based on a total dry weight of untreated input biomass inaccordance with a mixing molar ratio of an acid (HCl)-base (NaOH)mixture catalyst (a final concentration is 0.1 M) (A) and aconcentration of an acid-base mixture catalyst (mixed at a molar ratioof an acid: a base=2:1) (B). Pretreatment was performed at 190° C. inconditions of ramping for 3 minutes and then holding for 2 minutes, andan amount of solids loading was 10% (w/v). Enzymatic hydrolysis wasperformed using 15 FPU of cellulase (Cellic CTec2)/g glucan at 50° C.(pH 4.8) at 200 rpm for 50 hours.

As shown in FIG. 14, in the case of pretreatment with an acid-basemixture catalyst under an acidic condition, a superior saccharificationyield was observed compared to neutral and basic conditions (FIG. 14A),it can be seen that a concentration of an acid-base mixture catalystaffects a saccharification yield.

Next, in order to confirm stability of an acid-base mixture catalyst atnormal temperature, an acid-base mixture catalyst was incubated atnormal temperature for a predetermined time before pretreatment, andthen pretreatment of sugarcane bagasse and enzymatic hydrolysis wereperformed, and an insoluble solid recovery yield, a glucan recoveryyield, a lignin removal rate, and enzymatic digestibility were measuredin accordance with the incubation time of an acid-base mixture catalyst.Pretreatment was performed with a 0.1 M acid-base mixture catalyst (mixat a molar ratio of an acid: a base=2: 1) in a mini reactor at 190° C.in conditions of ramping for 3 minutes and then holding for 2 minutes,and an amount of solids loading was 10% (w/v). Enzymatic hydrolysis wasperformed using 15 FPU of cellulase (Cellic CTec2)/g glucan at 50° C.(pH 4.8) at 200 rpm for 50 hours.

As shown in FIG. 15, catalytic activity for pretreatment was almostidentical regardless of the incubation time of an acid-base mixturecatalyst. Therefore, it can be seen that characteristics of an acid-basemixture catalyst as catalyst for pretreatment are maintained at normaltemperature.

The following Table 2 shows results of composition analysis of sugarcanebagasse pretreated based on catalysts, wherein the acid-base mixturecatalyst was similar to HCl and an HCl-NaCl mixture catalyst in terms ofresults of pretreatment, but a product pretreated with an acid-basemixture catalyst showed a relatively high saccharification yield (FIG.16).

FIG. 16 shows enzymatic digestibility obtained through pretreatment ofsugarcane bagasse by using an acid-base mixture catalyst of 0.067 MHCl+0.033 M NaOH, 0.033 M HCl, 0.033 M NaCl, an acid-base mixturecatalyst of 0.033 M HCl+0.033 M NaCl, and water, wherein conditions ofpretreatment and enzymatic hydrolysis is the same as described above.

TABLE 2 Composition analysis of sugarcane bagasse pretreated withdifferent catalysts acid-base mixture catalyst NaCl (0.067 M (0.033 M) ±HCl ± 0.033 M HCl NaCl HCl untreated NaOH) (0.033 M) (0.033 M) (0.033 M)H₂O Component from insoluble solids (g per 100 g dry dried sugarcanebagasse before pretreatment) insoluble NA^(d) 62.7 ± 3.2 62.2 ± 0.6 86.6± 2.9  63.6 ± 2.4 87.6 ± 2.5 solid recovery yield glucan 40.9 ± 0.1 57.2± 1.6 57.8 ± 0.7 45.4 ± 1.9  58.5 ± 1.1 43.6 ± 1.2 xylan 26.5 ± 0.5 — —19.1 ± 0.8 — 19.6 ± 1.2 other  5.4 ± 0.1 — — — — — carbohydrates acid-24.0 ± 0.1 19.4 ± 0.1 19.3 ± 0.1 21.1 ± 0.1  19.7 ± 0.3 20.6 ± 0.5insoluble lignin acid-soluble  1.4 ± 0.1  1.3 ± 0.0  1.2 ± 0.0  1.4 ±0.0   1.2 ± 0.0  1.4 ± 0.0 lignin Component from dissolved solids (g per100 g dry dried sugarcane bagasse before pretreatment) glucose NA^(d) 4.3 ± 0.0  3.1 ± 0.0  0.9 ± 0.0   3.8 ± 0.1  1.0 ± 0.0 hemicelluloseNA^(d) 21.1 ± 0.3 18.4 ± 0.3  6.6 ± 0.2  21.4 ± 0.4  7.0 ± 0.3monomer^(c) acetic acid NA^(d)  2.8 ± 0.0  2.4 ± 0.0  0.1 ± 0.0   2.6 ±0.0  0.1 ± 0.0 formic acid NA^(d)  0.2 ± 0.0  0.1 ± 0.0 —   0.2 ± 0.0 —furfural NA^(d)  1.8 ± 0.1  1.3 ± 0.1   0.2 ± 00.0   1.4 ± 0.0  0.2 ±0.0 ^(a)Pretreatment conditions: ramping at 190° C. for 3 minutes,holding for 2 minutes, and solids loading in an amount of 10% (w/v)^(b)Experimental data is represented as means ± standard deviations^(c)xylose, galactose, and mannose are included in a liquid fraction^(d)NA: untreated

The present invention is a technique applicable to the field ofbio-ethanol preparation.

What is claimed is:
 1. A method for pretreating lignocellulosic biomasscomprising: reacting an aqueous acid-base mixture catalyst having a pHvalue of 1 to 4 with lignocellulosic biomass at 100 to 200° C. for 60seconds to 2 hours; wherein the acid is selected from the groupconsisting of sulfuric acid, maleic acid, hydrochloric acid, nitricacid, phosphoric acid, carbonic acid, formic acid, acetic acid,hydrofluoric acid, oxalic acid, citric acid, and mixtures of two or morethereof; wherein the base is selected from the group consisting ofsodium hydroxide, potassium hydroxide, calcium hydroxide, bariumhydroxide, ammonium hydroxide, calcium carbonate, potassium carbonate,ammonia, and mixtures of two or more thereof; and wherein said acid-basemixture catalyst has an acid:base molar ratio of 4:1, and has a totalconcentration of 0.05 M to 0.5 M.
 2. The method for pretreatinglignocellulo sic biomass according to claim 1, wherein thelignocellulosic biomass is mixed in a concentration of 4 g to 20 g per100 mL of the acid-base mixture catalyst.
 3. The method for pretreatinglignocellulosic biomass according to claim 1, wherein thelignocellulosic biomass is selected from rice straw, giant miscanthus,sugarcane bagasse, corn byproducts, switchgrass, poplar, oil palmbyproducts, or oak.